RNA-protein interaction. An analysis with RNA oligonucleotides of the recognition by alpha-sarcin of a ribosomal domain critical for function.
alpha-Sarcin is a cytotoxic protein that inactivates ribosomes by hydrolyzing a single phosphodiester bond on the 3' side of G-4325 in eukaryotic 28 S rRNA. We have examined the requirements for the recognition by alpha-sarcin of this domain using a synthetic oligoribonucleotide (35-mer) that reproduces the sequence and, we presume, the secondary structure (a stem, a bulged nucleotide, and a loop) at the site of modification. The wild type structure and a large number of variants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. Recognition of the substrate is strongly favored by a G at the position that corresponds to 4325. There is an absolute requirement for a helical stem; however, it can be reduced from the 7 base pairs in the natural structure to 3 without loss of specificity. The nature of the base pairs in the stem modifies but does not abolish recognition; whereas, the bulged nucleotide does not contribute to identification. Cleavage is materially affected by altering the nucleotides in the universal sequence surrounding G-4325 and changing the position in the loop of the tetranucleotide GAG(sarcin)A leads to loss of recognition by the toxin. We propose that the alpha-sarcin domain RNA participates in elongation factor catalyzed binding of aminoacyl-tRNA and of translocation; that translocation is driven by transitions in the structure of the alpha-sarcin domain RNA initiated by the binding of the factors, or the hydrolysis of GTP, or both; and that to toxin inactivates the ribosomes by preventing this transition.
Endo
Y
Y
Department of Biochemistry, Yamanashi Medical College, Japan.
Glück
A
A
Chan
Y L
YL
Tsurugi
K
K
Wool
I G
IG
D000241:Adenosine; D001230:Aspergillus / Q000378:metabolism; D001482:Base Composition; D001483:Base Sequence; D004722:Endoribonucleases; D005656:Fungal Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005656:Fungal Proteins; D009843:Oligoribonucleotides; D011500:Protein Synthesis Inhibitors; D012339:RNA, Ribosomal, 28S; C014801:alpha-sarcin; D004722:Endoribonucleases; D000241:Adenosine